Organophosphorus (OP) insecticides are the principal cause of pesticide-related human poisonings. Recent restrictions on the use of chlorpyrifos and diazinon reflect continuing concern for human health, particularly children. The two principal targets of OP toxicant action are acetylcholinesterase (AChE) for acute toxicity and neuropathy target esterase (NTE) for OP-induced delayed neuropathy (OPIDN). This research focuses on noncholinergic effects, based in part on findings with collaborators on nullizygous AChE-knockout mice (AChE -/-) and heterozygous NTE-knockout mice (NTE ). AChE -/- mice are extremely sensitive to chlorpyrifos oxen (CPO) (intraperitoneal LD50 0.45 mg/kg) compared with their wild-type littermates (LD30 3 mg/kg), establishing the importance of an unidentified non-AChE target for acute lethality. The first specific aim is to identify this non-AChE target in mammals. Mouse brain proteins from AChE -/- and wild-type mice will be radiolabeled in vitro and ex vivo with [3H-ethyl]CPO. Differential protein labeling in the AChE -/- and wild-type mice with very low levels of [3H]CPO will allow selection, purification and identification of the candidate alternate target of OP acute poisoning. The second aim is to establish the function of NTE in OPIDN using mice as the model. The NTE mice have a 40% reduction in NTE activity, making them ideal for toxicological investigations. The studies will focus on the association of NTE levels with behavioral and neuropathological changes, the sensitivity of NTE and wild-type mice to ethyl octylphosphonofluoridate and other OP delayed toxicants, validation of the mouse model for OPIDN and the mechanism by which lowered NTE induces hyperactivity and delayed toxicity. The third aim is to define the mechanisms of toxicity from disruption of signal transduction pathways, and more specifically those mediated by lysophospholipase, diacylglycerol lipase, and the muscarinic acetylcholine receptor. The last goal is to define the mechanisms and significance of two secondary targets of OP pesticides: altered endocannabinoid action by OP phosphorylation at a nucleophilic site coupled to the cannabinoid receptor-1 (CB1) agonist site; kynurenine formamidase (KFase) structure and function relative to teratogenesis and diazinon oxen action in the brain. Knowledge gained on OP pesticide toxicology is also applicable to OP nerve gases as chemical warfare and terrorism agents.